1. Field of the Invention
The present invention relates to the electrophoretic separation of molecules, in particular proteins and, more specifically, to the electrophoretic separation in two dimensions, a first separation using an isoelectric focusing technique along a first direction and a second separation according to the molecular weight of the molecules along a second direction.
2. Description of Prior Art
The study of life sciences involves classification and characterisation of a variety of biomolecules. The study of proteins is ubiquitous in life sciences and many techniques have been used to isolate, separate and characterise proteins from a variety of sources. Established techniques for protein separation included electrophoresis and liquid chromatography. Traditionally electrophoretic techniques have been used to separate and characterise proteins. Proteins, in common with the majority of biomolecules are charged or can be made to be charged by defining the media in which they are solved. Consequently they will move, in solution, under the influence of an electric field with a velocity which is dependent on the charge to mass ratio of the protein; when the molecule has no charge it has no mobility.
Two traditional electrophoretic techniques for protein separation are isoelectric focusing (IEF) and SDS-PAGE.
IEF will separate proteins on the basis of their isoelectric point. Most proteins carry a number of charged or chargeable side chains in addition to the N- and C-terminal moieties which are also capable of carrying a charge. Depending on the pH of the buffer in which the protein is contained, these chargeable groups will carry a 0 to +1 charge if it is an amine function, and -1 to 0 charge if it is an acid group. Since the degree of ionisation will also depend on local environment, this will result in a number of different degrees of charged states over the entire protein. At a particular and idiosyncratic pH, the mixture of positive and negative charges will balance and the protein will have a net charge of 0. This property can be harnessed to separate proteins on the basis of the pH at which their net charge is zero. This pH is referred to as the isoelectric point or pI of the protein.
IEF is operated by constructing a pH gradient between two electrodes with the highest pH at the negative electrode (cathode) and lowest pH at the positive electrode (anode). A pH gradient may be created using a complex mixture of chemicals called ampholytes. These will arrange themselves between the anode and cathode such that they create a gradient of increasing pH from the anode to the cathode. When proteins are introduced into this system, their charge will depend on the pH of the environment in which they find themselves. If the environmental pH is lower than the proteins pi, then it will have a net positive charge and will migrate towards the negative electrode. In this direction, the pH increases and the protein's net charge will become zero when the local pH is the same as it's pI. Any further movement via diffusion in the direction of the negative electrode will expose the protein to a pH higher than its pI and at this point its net charge will become negative and vice versa. The protein will then migrate back towards the positive electrode until it focuses into the pH region equal to its pI. In this way, the proteins will focus in different areas depending upon their isoelectric points and this technique allows separation on the basis of pI. If a standard mixture of proteins is used with known pI's, then the sample proteins can be characterised by calculating their pI relative to the standards. Such separations are traditionally carried out in supportive media, i.e., gels or in a capillary format.
Such techniques for performing a separation on the basis of isoelectric focusing are disclosed in U.S. Pat. No. 5,320,727, for example.
A second technique for separating proteins is to separate them on the basis of their molecular weight. This can also be achieved using electrophoretic phenomena. In this case, the proteins are incubated with a chemical (sodium dodecyl sulphate (SDS)) which has a 12-carbon tail attached to a negatively charged sulphonic acid group. The C12 chain is hydrophobic and will associate with hydrophobic regions on the protein so that the negative head is projected outwards from the protein. This is usually achieved after the protein has been denatured and the resulting protein-SDS structure is linear and negatively charged. Proteins will take up SDS molecules with a relatively constant ration of 1:1,4 (protein:SDS). Therefore, these structures will have equivalent charge to mass ratios. This means that the SDS-protein structures have similar mobilities. SDS-proteins may be separated by causing them to migrate through a sieving structure usually created by making a cross-linked gel or a solution of entangled polymers. In both cases, small SDS-protein molecules will travel faster than larger SDS-protein molecules and any mixture of these will therefore separate according to molecular weight. If a standard solution of proteins of known molecular weight is used, then the molecular weight of sample proteins may be determined by comparing their migration position relative to that of the known standard. These two techniques can be combined to provide a separation of a complex mixture of proteins firstly in one dimension by IEF and secondly by SDS-protein sieving. The combination of two separation selectivities in orthogonal directions provides a powerful way of separating very complex mixtures or of characterising a protein product. This technique is becoming widely used in the field of the separation of proteins.
One combined method for high-resolution two-dimensional electrophoresis is disclosed in U.S. Pat. No. 5,407,546. This prior art method comprises the steps of carrying out at first, on a gel base, a first separation process in a first direction by isoelectric focusing and performing thereafter a second separation process in a direction vertical to the first direction by a technique different from that of the first separation. In accordance with the method taught in this document, a predetermined quantity of an IEF mixture is dispensed upon a strip-like fleece positioned on the marginal area of a dry gel layer in order to produce an IEF plateau. At the end of a predetermined period of time, the fleece is pulled-off using a pair of tweezers such that the IEF plateau remains in the dry gel layer. Thereafter, two electrophoresis processes are carried out simultaneously using a common central cathode and two anodes which are provided in two end portions of the dry gel layer.
The gels used to support the separations are generally made by the user, although these can be purchased pre-cast. These are constructed in a planar format by mixing a polymer with a certain percentage of a cross-linker. The higher percentage of cross-linker the smaller is the pore size created. Small pore sizes are of more use for small molecules and provide better differentiation or resolution between proteins of similar molecular weights. It is often useful to cast a gel where the pore size decreases linearly in one direction. This is referred to as a gradient gel and is capable of providing better resolutions of a wide range of molecular weights.
After separation in either one or two dimensions, the proteins can be reacted with a dye, such as Coummassie Blue, which will stain the protein and allow its detection. Alternatively, the gel spot containing the protein can be cut out from the gel and the protein digested with an enzyme such as trypsin to give smaller fragments. The size and number of these fragments is dependent on the amino acid sequence of the proteins and, therefore, can be defining and idiosyncratic. Another alternative is that after separation on the gel, the proteins can be transferred via electro-blotting onto a membrane. On this membrane, the protein can be subjected to a variety of tests such as immunostaining, or digested on the membrane with e.g., trypsin. The fragments resulting from digestion on the membrane can be introduced directly into a mass spectrometer which will give the molecular weights of the fragments. The number and molecular weight of the protein can be enough to identify it when compared to a protein database. Alternatively, MS/MS (MS=mass spectroscopy) can be performed in order to sequence the peptides and, therefore, provide their amino acid sequence.
The manufacturing and use of a different structure which will separate molecules under the influence of an electric field in a sieving matrix is disclosed in U.S. Pat. No. 5,427,663. According to this document, molecules which are large pieces of DNA are separated, wherein the sieving matrix is an array of physical posts which create a physical matrix which must be manoeuvred by the DNA molecules as they are driven by the electric field across the planar structure. U.S. Pat. No. 5,427,663 discloses a sorting apparatus and method for fractionating and simultaneously viewing individual microstructures and macromolecules, such as DNA molecules, proteins and polymers. In accordance with U.S. Pat. No. 5,427,663, a substrate having a shallow receptacle located on a side thereof is provided. An array of obstacles upstanding from the floor of the receptacles is provided in order to interact with the microstructures to partially hinder the migration thereof in a migration direction through the receptacle. Electrodes for generating an electric field in the fluid medium in order to induce the migration of the microstructures are provided on two sides of the receptacle. The receptacle is covered by a transparent cover, such that the height of the receptacle is commensurate with the size of the microstructures to be sorted in the sorting apparatus. Thus, when the microstructures are caused to migrate in the fluid medium through the receptacle, the microstructures do so in essentially a single layer. This is an analogous mechanism to the sieving of SDS-proteins using cross-linked gels or entangled polymers.
General techniques for the fabrication of microstructures with high aspect ratios and great structural heights by synchrotron radiation lithography, galvanoforming and plastic moulding (LIGA process) are disclosed by E. W. Becker, et al., Microelectronic Engineering 4 (1986), pages 35 to 56.
H. Becker, et al., J. Micromech. Microeng. 8 (1998), pages 24 to 28, teaches a planar quarz chip with sub-micron channels for two-dimensional capillary electrophoresis applications. In this chip, the first separation dimension consists of a single channel, whereas the second separation dimension consists of an array of 500 channels. The channels are fabricated using reactive iron etching under maximum anisotropic conditions, yielding an aspect ratio of up to 5 for the narrow channels along which the second dimension separation is performed.